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Introduction
G-quadruplexes are four single-stranded structures that are formed by nucleic acid sequences rich in guanine. These strcutures are made of a square arrangement of guanines, known as a tetrad, and is stabilized by Hoogsteen face hydrogen bonding. A monovalent cation (either sodium or potassium) in the center of the tetrads also stabilizes these structures. The directions of the strands of the tetrads determine whether the structure is parallel or antiparallel.[3]

The G-quadruplex structures are formed by four single strands of telomeric DNA. Daunomycin binds to the DNA only when DNA is in the G-Quadruplex form and does so by intercalating. In intercalating three molecules of daunomycin fit in between two base pairs and forms hydrogen bonds with the phosphates of the DNA backbone.[2]

For the telomerase enzyme to catalaze synthesis of new telomeric DNA the 3' terminus of the primer DNA needs to be single strand to properly bind with the RNA template of the enzyme. The replication of these telomeric DNA repeats elongates the telomeric DNA and is what prevents apoptosis (cell death) in cancer cells. When the single stranded telomeric DNA is folded into the four-stranded G-Quadruplex it inhibits the telomerase from synthesizing new telomeric DNA. Compounds that promote the formation of the G-Quadruplex are therefore inhibitors of the telomerase enzyme.[2] Telomerase is a tumor-selective target in chemotherapy. The inhibition of telomerase is important because it is over-expressed in about 85% of tumor cells, which leads to the prevention of natural shortening of the telomere and rapid cell growth.[1] This is unlike normal cells, which shorten the telomere sequence after each cell division and leads to the stoppage of cell division (senescence) and then finally apoptosis.[2]

With telomerase inhibition, cancer cells can be tuned back for natural cell death. There must be high binding selectivity towards the intramolecular G-quadruplex structure of telomere over a DNA duplex structure in order to have a G-quadruplex stabilizing agent. [1]

Structure
Telomeric DNA is mostly double-helical, however; a single strand extends at the 3' terminus. This strand can interact with other strands of Telomeric DNA to form a four stranded quadruplex structure. These quadruplex structures consist of stacked guanine-tetrads that are 3.35 A apart with a sodium ion between each G-tetrad. Crystallographic studies have revealed that all strands are parallel in the intermolecular quadruplex formed.[1]

These guanine-tetrads are the repeat motif common to all G-quadrupexes. The G-tetrads consists of a plane of four guanine bases stabilized by hydrogen bonding of the Hoogsteen face. Within any given G-quadruplex there are three to four G-quartets stacked one on top of the other and are further stabilized by pi-pi non-bonded attractive interactions. [4]

The G-quadruplex (G4) has many significant structural differences from double stranded DNA. The two main different features are the four equally sized grooves and a channel of negative electrostatic potential running through the center of the planes of the G-quartets, allowing sodium cations to coordinate between the planes .[2]

Binding Sites
The first crystal structure of a drug (daunomycin) bound to a parallel-stranded intermolecular telomeric G4 quadruplex.[1]

The daunomycin layer are tightly packed onto the end of the quadruplex where the daunosamine sugars form H-bonding interactions and/or van der Waals contacts with three of the four quadruplex grooves.

The daunosamine sugar of the first daunomycin is wedged tightly into its groove, and its cationic amine substituent H-bonds to the phosphate oxygens on both sides of the grooves with a distance of 2.81 Å and 2.79 Å. (Binding Site 1) The sugar of the second daunomycin is not as deep in its groove, and H-bonds are found from its cationic amine and exocyclic OH groups to phosphate oxygens on just one side of the groove with a distance of 2.68 Å. (Binding Site 2) Lastly there are no direct H-bonds between the daunosamine of the third daunomycin and the quadruplex. [1]

Additional Features
Telomeres shorten in normal somatic cells with each round of replication; this is a result of DNA polymerase's inability to fully replicate the ends of the DNA strands. Eventually the telomere length reaches a critical point that triggers a senescent state at which the cell ceases to replicate, eventually dying. Tumor cells do not suffer this though; they have short, stable telomeres that are replicated by a telomerase enzyme unique to tumor cells. These replicated telomeres are responsible for the immortalization of human cells, leading to tumorigenesis. Inhibition of telomerase leads to natural telomere shortening and eventually cell apoptosis, making telomerase inhibitors an attractive anti-cancer therapy option.[2]

Compounds that have been shown to inhibit the telomerase enzyme are tricyclic aromatic chromophores. Some examples are anthraquinones, fluorenones, and acridines; daunomycin is an acridine. Compounds that show the highest activity has come by substituting the side chains with groups having amidoalkylamino character. The exploration into these synthetic molecules that stabilize the G4 complex have been investigated as potential anti-cancer drugs. Most conventional anti-cancer drugs demonstrate mutual selectivity towards the duplex and quadruplex forms. The distinct structural differences between G2 and G4 allow for the development of new anti-cancer drugs, that are more effective than existing G2 selective drugs.

Credits
Introduction: Joe Perito/Paul Breslin

Overall Structure: Lyes Khendek/William Rowley/Ashley Rivera

Drug Binding Sites: Lyes Khendek

Additional Features: WiIliam Rowley/Paul Breslin